HAb18G/CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner
BMC Cell Biology
RHesAeabrch1ar8ticGle /CD147 cell-cell contacts confer resistance of a HEK293 subpopulation to anoikis in an E-cadherin-dependent manner
Xiao-Kui Ma 1
Li Wang 1
Yu Li 1
Xiang-Ming Yang 1
Pu Zhao 1
Ping Zhu 0
Ling Li 1
Zhi-Nan Chen 1
0 Department of Clinical Immunology in Xijing Hospital, Fourth Military Medical University , 17 Changle West Road, Xi'an 710032 , PR China
1 Cell Engineering Research Center & Department of Cell Biology, National Key Discipline of Cell Biology, State Key Laboratory of Cancer Biology, Fourth Military Medical University , 17 Changle West Road, Xi'an 710032 , PR China
Background: Acquisition of resistance to "anoikis" facilitates the survival of cells under independent matrix-deficient conditions, such as cells in tumor progression and the production of suspension culture cells for biomedical engineering. There is evidence suggesting that CD147, an adhesion molecule associated with survival of cells in tumor metastasis and cell-cell contacts, plays an important role in resistance to anoikis. However, information regarding the functions of CD147 in mediating cell-cell contacts and anoikis-resistance remains limited and even self-contradictory. Results: An anoikis-resistant clone (HEK293ar), derived from anoikis-sensitive parental Human Embryonic Kidney 293 cells, survived anoikis by the formation of cell-cell contacts. The expression of HAb18G/CD147 (a member of the CD147 family) was upregulated and the protein was located at cell-cell junctions. Upregulation of HAb18G/CD147 in suspended HEK293ar cells suppressed anoikis by mediating the formation of cell-cell adhesions. Anoikis resistance in HEK293ar cells also required E-cadherin-mediated cell-cell contacts. Knock-down of HAb18G/CD147 and E-cadherin inhibited cell-cell contacts formation and increased anoikis sensitivity respectively. When HAb18G/CD147 was downregulated, E-cadherin expression in HEK293ar cells was significantly suppressed; however, knockdown of Ecadherin by E-cadherin siRNA or blocking of E-cadherin binding activity with a specific antibody and EDTA had no significant effect on HAb18G/CD147 expression. Finally, pretreatment with LY294002, a phosphoinositide 3-kinase (PI3K/AKT) inhibitor, disrupted cell-cell contacts and decreased cell number, but this was not the case in cells treated with the extracellular signal-regulated kinase (ERK) inhibitor PD98059. Conclusions: Our results provide new evidence that HAb18G/CD147-mediated cell-cell contact confers anoikis resistance in an E-cadherin-dependent manner; and cell-cell contact mediated resistance to anoikis implicates PI3K pathway in a highly relevant cell model (HEK293ar). Understanding of the role of HAb18G/CD147 cell-cell contacts in anoikis resistance may help in understanding the survival of cells in anchorage-independent growth, such as cells in tumor metastasis and suspension culture produced for biomedical engineering. Our results also contribute to a better understanding of the biology of HEK293 cell spheroids, a major workhorse for producing human therapeutic agents and viral vaccines.
CD147, an extracellular matrix metalloproteinase inducer
(also known as EMMPRIN, basigin, M6), is a plasma
membrane-bound glycoprotein that functions as an
adhesion molecule. It is expressed at high levels on a
variety of malignant human cancers and some immortalized
cell lines. Our laboratory previously identified a novel
hepatoma associated antigen named HAb18G, which was
obtained by cloning a human hepato-cellular carcinoma
(HCC) cDNA library and screening with the
anti-hepatoma monoclonal antibody HAb18 . The nucleotide
acid and amino acid sequences of HAb18G are identical
to those of CD147 . HAb18G/CD147 was highly
expressed by HCC cells and tissues, and increased
HAb18G/CD147 expression stimulated both the growth
and invasiveness of HCC cells, much as CD147 functions
in other cancer cells [3-5].
The acquisition of resistance to anoikis, a form of
apoptosis triggered by loss or alteration of cell-cell or
cellmatrix anchorage, is critical for the survival of cells in
tumor progression and suspension growth used in
engineering. Resistance to anoikis is emerging as a hallmark
of metastatic cancer cells, important in tumor
progression because it increases survival times in the absence of
cell anchorage, facilitating migration and reattachment,
and therefore increasing the probability of metastasis .
Furthermore, acquisition of anoikis resistance is required
for cells used in engineering during adaptation to
suspension culture and spheroid growth.
More recently, CD147 has been reported as an anoikis
suppressor, promoting anchorage-independent growth
by stimulating hyaluronan production  and regulating
the anoikis signal pathway by upregulating Bim .
However, it is not clear whether the role of CD147 in anoikis
resistance is related to cell adhesion, which is a basic
function of this molecule in addition to its role in
stimulating matrix metalloproteinase (MMP) secretion .
Different bioactive epitopes of CD147 involved in
regulating cell adhesion have been identified . CD147 has
also been reported to participate in forming compacted
cell aggregates by regulating fibronectin matrix assembly
 and cell-cell adhesion . The binding of CD147
mAb to CD147 may mimic natural ligand-receptor
binding and induce homotypic U937 monocytic cell
aggregation via the LFA-1/ICAM-1 pathway . In contrast,
Cho reported that antibodies to CD147 are potent
inhibitors of homotypic U937 aggregation induced via CD98
ligation . Because the establishment/maintenance of
cell-cell contacts is considered an important
environmental condition for physiological resistance to anoikis, we
hypothesized that CD147 may confer anoikis resistance
by mediating cell-cell adhesion. Unfortunately, direct
evidence for the role of CD147 in mediating cell-cell
contacts and anoikis resistance is very limited and even
selfcontradictory. Furthermore, it is not clear whether
CD147 is directly involved in cell adhesion either as an
adhesion signal transmitting molecule or a regulator.
The aim here was to explore whether HAb18G/CD147
is involved in forming cell-cell contacts, and whether this
contributed to its role in regulating anoikis resistance. A
transformed cell line, Human Embryonic Kidney (HEK)
293, was chosen as the model because our previous
results confirmed that these cells express HAb18G/
CD147 . We also acquired an anoikis-resistant
subpopulation (HEK293ar) from the anoikis-sensitive
parental HEK293 cells. Together, these two cell types provide
an ideal model for exploring the role of HAb18G/CD147
as a cell-cell adhesion molecule preventing anoikis. Our
results show that HAb18G/CD147 cell-cell contacts
confer anoikis resistance and this function also involves
Ecadherin expression in HEK293ar cell spheroids.
Anoikis-resistant HEK293ar cells survive anoikis by the
formation of cell-cell contacts
To explore the role of cell-cell contacts in preventing
anoikis, we generated a subpopulation (HEK293ar) with
an anoikis-resistant phenotype derived from HEK293
cells using sequential cycles of adhesion and suspension
culture . Upon detachment, the apoptotic ratio in
HEK293ar cells was lower than in the parental cells, as
shown by quantifying the proportions in sub-G1 by flow
cytometry (6.97 3.1% versus 37.3 2.3%, data not
shown). When HEK293ar cells were cultured in
suspension for 1, 10 and 30 days, the mean sub-G1 proportions
were 8.7, 11.7, and 15.1%, respectively, with little change
in the apoptotic ratio (Fig. 1A). Moreover, this
subpopulation maintained the anoikis resistance phenotype in
vitro for 6 months (data not shown). Additionally, after
~6-10 h suspension culture, the HEK293ar cells
spontaneously and gradually formed cell-cell contacts and
generated multi-cellular spheroids that could still grow as
adhesion cultures, whereas the parental HEK293 cells did
not (Fig. 1B). Ultrastructural examination demonstrated
that the suspended spheroids comprised many single
cells. In each spheroid, chromatin masses of moderate
electron density were dispersed in the nuclei, and the
nucleoli were conspicuous (Fig. 1C, arrowed). Cells
within the multi-cellular spheroids adhered laterally to
each other through diverse specialized intercellular
junctions (Fig. 1D, arrowhead). Pretreatment with EDTA
solution to disrupt calcium-dependent cell-cell contacts
also disrupted the adhesions and caused massive DNA
fragmentation in HEK293ar cells, as shown by TUNEL
assay (Fig. 1E). Interestingly, single cells detached from
the aggregates gave more intense TUNEL staining (Fig.
1E, arrowhead). In contrast, TUNEL assays and
ultrastructural examination demonstrated that HEK293 cells
clearly underwent anoikis (data not shown). It was
previously reported that cell-cell contacts prevent anoikis in
primary human colonic epithelial cells . The present
results also suggest that adjacent HEK293ar epithelial
cells form cell-cell contacts, suppressing anoikis under
HAb18G/CD147 promotes HEK293ar cell survival in
suspension by mediating cell-cell contacts
We next explored whether HAb18G/CD147 was involved
in HEK293ar cell spheroid survival by mediating cell-cell
contacts. HAb18G/CD147 expression was compared
Figure 1 Anoikis-resistant HEK293ar cells survives anoikis through cell-cell contacts. An anoikis-resistant HEK293 cell subpopulation (HEK293ar)
was obtained by sequential cycles of adhesion and suspension culture. A. Anoikis in HEK293ar cells in suspension culture at different time points. Flow
cytometry (histogram) shows that the apoptosis rate changed little during suspension culture of HEK293ar. The x and y-axes indicate the size of DNA
and the number of cells counted, respectively. FL3-H, a standard term for flow cytometry, represents measurement of the fluorescence intensity of
propidium iodide (PI) at a super-red wavelength (670 nm). The results are means S.D. (n = 4-6). B. The morphology of HEK293ar and parental HEK293
cells in suspension and adhesion culture revealed by microscopy (n = 4-10). Magnification: 200. C. TEM of a multicellular HEK293ar spheroid.
Chromatin masses of moderate electron density are dispersed in the nuclei and nucleoli are conspicuous (arrowed). The cells were observed in 4-6
independent sections. Bar, 2 m. D. A junctional complex with thickened membranes (arrowed) in suspended HEK293ar cells viewed under SEM. Bar, 50
nm. The cells were viewed in 4-10 independent sections (at least 100 cells/section). E. EDTA (10 mmol/l) treatment disrupted the cell-cell contacts and
resulted in massive apoptosis as determined by TUNEL assay. Arrowheads show that single cells detached from the aggregates give more intense
TUNEL staining. Magnification: 200. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/section).
between HEK293ar and HEK293 cells in suspension
culture. HEK293ar cells expressed significantly more
HAb18G/CD147 than the parental cells after 24 h (Fig.
2A). Positive immunofluorescence staining for CD147
was restricted to the cell-cell contacts of HEK293ar (Fig.
2B, merge, arrowhead). These results may imply a
correlation between HAb18G/CD147expression and cell-cell
contacts-directed anoikis resistance, as the level of
HAb18G/CD147 expression was similar in both cell types
in adhesion culture (data not shown). To obtain further
insight into this correlation, HEK293ar cells were treated
with HAb18G/CD147 siRNA in adhesion culture, and
apoptosis and cell-cell contacts formation were
determined in suspension culture. When HAb18G/CD147
expression was reduced by ~70% (Figs. 2C, D; **p < 0.01),
HEK293ar cells underwent anoikis with a sub-G1
proportion of 37.9 2.1% (Fig. 2E). Cell-cell contacts formation
was totally inhibited after suspension, whereas the
control cells formed multicellular aggregates after as little as
12 h in suspension (Fig. 2F). These results indicate that
HAb18G/CD147 confers anoikis resistance of HEK293ar
cells to anoikis by mediating cell-cell contacts formation.
Figure 2 HAb18G/CD147 promotes HEK293ar cells survival by mediating cell-cell contacts. A. Western blotting to reveal HAb18G/CD147
expression in HEK293ar and parental HEK293 cells in suspension culture. Two major forms of HAb18G/CD147 (43-66 and 35 kDa) were analyzed.
-Tubulin was used as a loading control (n = 4-6). B. Immunofluorescence of HAb18G/CD147 under laser scanning confocal microscope (Olympus FV1000,
Tokyo, Japan; n = 4-10). The nuclei were counterstained with DAPI (4',6-diamidino-2-phenylindole). Magnification: 1200. C. Western blotting to reveal
HAb18G/CD147 expression in HEK293ar cells in HAb18G/CD147 RNA interference (n = 4-6). -Tubulin was used as a loading control. D. Comparison
of the gray scale ratio of HAb18G/CD147/-tubulin in HAb18G/CD147 RNA interference (n = 4-6). ** p < 0.01, siRNA versus scrambled. E. Flow
cytometric quantification of anoikis after siRNA treatment (n = 4-6). Anoikis was determined as described in Fig. 1A. F. Cell-cell contacts formation with
time after treatment of targeted HAb18G/CD147-siRNA under the phase-contrast microscope (n = 4-6). Magnification: 400. Each value represents
the mean SD of at least triplicate determinations. Results are the representative of three similar experiments.
Cell-cell contact-directed survival in suspension involves
Upon detachment, anchorage-independent Ewing
sarcoma cells suppressed anoikis through a pathway
involving E-cadherin-dependent cell-cell adhesion . Thus
we explored whether E-cadherin was also involved in
suppressing anoikis by mediating cell-cell adhesion in
HEK293ar cells. As anticipated, increased E-cadherin
expression was confirmed in HEK293ar cells after 24 h
suspension culture (Figs. 3A, B, *p < 0.05), and positive
staining was partly located around the cell-cell contacts
(Fig. 3C). Also, no significant expression of E-cadherin in
completely suspended parental HEK293 cells or in the
initially suspended HEK293ar cells was found by
immunofluorescence. A reasonable explanation could be that
Ecadherin expression decreases dramatically upon cell-cell
detachment . In addition, a 70-80% reduction of
Ecadherin expression by siRNA (Figs. 3D, E; ** p < 0.01), as
determined by western blotting, totally inhibited cell-cell
contacts formation (Fig. 3F) and decreased the degree of
cell aggregation with 70-80%, scored as described in
Methods. Knockdown of E-cadherin led to a marked
increase in the mean sub-G1 proportion and a little
shifted peaks in Flow cytometric histogram (Fig. 3G, * p <
0.05), and it also decreased the cellular DNA content via
fluorescent dye binding determined with a CyQUANT
Figure 3 Cell-cell contact-directed HEK293ar cell survival requires the involvement of E-cadherin. A. Increased E-cadherin expression in
HEK293ar cells revealed by western blotting (n = 4-6). B. Comparison of the gray scale ratio of E-cadherin/-tubulin between HEK293 and HEK293ar
cells (n = 4-6). * p < 0.05, HEK293 versus HEK293ar. C. E-cadherin expression in HEK293ar and HEK293 cells revealed by immunofluorescence under
laser scanning confocal microscope (Olympus FV1000, Tokyo, Japan; n = 4-10). Magnification: 1600. D. Western blotting to reveal E-cadherin
expression in E-cadherin RNA interference (n = 4-6). E. Comparison of the gray scale ratio of E-cadherin/-tubulin in E-cadherin RNA interference (n = 4-6).
** p < 0.01, siRNA versus scrambled. F. The effect of E-cadherin RNA interference on the cell-cell contacts formation. Magnification: 1000. The nuclei
were counterstained with DAPI. The cells were viewed in 4-6 independent sections (at least 300 cells/section). G. Flow cytometric quantification of
anoikis after E-cadherin siRNA-treatment (n = 4-6). H. The effect of E-cadherein knockdown on the cell number of HEK293ar in suspension. * p < 0.05,
siRNA versus scrambled. The quantification of relative cell number was done with CyQUANT NF Cell Proliferation Assay Kit. This kit measures the
cellular DNA content via fluorescent dye binding, which is closely proportional to cell number (n = 4-10). Each value represents the mean SD of triplicate
determinations. Results are the representative of three similar experiments.
NF Cell Proliferation Assay Kit (Fig. 3H; *p < 0.05), which
is closely proportional to cell number. These results
suggest that cell-cell contacts formation and anoikis
resistance in HEK293ar cells may also be mediated by
HAb18G/CD147 mediates cell-cell contacts and anoikis
resistance through E-cadherin cell-cell contacts
As HAb18G/CD147 and E-cadherin are both related to
cell-cell contact-directed survival of HEK293ar cells in
suspension, they may be functional connected. To test
this hypothesis, we investigated the relationship between
these two molecules by RNAi. Figs. 4A, B and 4C show
that E-cadherin expression decreased dramatically in
HAb18G/CD147 siRNA-treated HEK293ar cells
according to immunofluorescence staining density (Figs. 4A,
analyzed by Image Pro Plus 6.0 3-DS software, **p < 0.01,
data not shown) and western blotting (Fig.4C, **p < 0.01).
Interestingly, with time after HAb18G/CD147 siRNA
treatment, cell-cell contacts and spheroids gradually
disrupted (Fig. 4A). In contrast, blocking of E-cadherin
binding by an anti-E-cadherin and inhibition of cell-cell
contacts with EDTA showed no significant effect on
HAb18G/CD147 expression at the single cell level
according to immunofluorescence staining (Fig. 5A;
fluorescence density was analyzed by Image Pro Plus 6.0
3DS, **p < 0.01, data not shown), although the degree of
cell aggregation was decreased in suspension culture (Fig.
5B, **p < 0.01). Furthermore, treatment of HEK293ar cells
with E-cadherin siRNA under adhesion conditions did
not alter the level of HAb18G/CD147 expression of
HEK293ar in suspension (Figs. 5C, D, ** p < 0.01; Fig. 5E,
p > 0.05). Together, these results indicate that the effect of
HAb18G/CD147 on cell-cell adhesion and anoikis
suppression is mediated by E-cadherin.
The Ras-ERK1/2 or PI3K/AKT pathway is involved in
Ecadherin or HAb18G/CD147-mediated cell adhesion and
survival [18-20]. To determine the situation, we
investigated whether these two pathways were involved in
cellcell contact-directed survival in suspension. HEK293ar
spheroids were treated with LY294002 (a specific
competitive PI3K inhibitor) or PD98059 (an ERK inhibitor).
As shown in Figs. 6A, B, LY294002 (20-50 mol/l)
inhibited the cell-cell contacts formation and decreased the
degree of cell aggregation in a dose-dependent manner,
but not PD98059. TUNEL assay showed that the
LY294002 (50 mol/l) treatment resulted in positive
staining of TUNEL in HEK293ar cells (Fig. 6C,
arrowhead), but PD98059 (50 mol/l) did not. Additionally,
LY294002 (20-50 mol/l) decreased relative fluorescent
intensity indicating the cellular DNA content in a
dosedependent manner (Newman-Keuls Multiple
Comparison Test, **p < 0.01,0 mol/l versus 20 mol/l; Fig. 6D)
and this result indicated that LY294002 treatment
decreased the cell number of HEK293ar in suspension in
a dose-dependent manner, as the cellular DNA content
via fluorescent dye binding is closely proportional to cell
number based on the instruction of the assay kit
(described in Methods). In contrast, PD98059 did not
significantly affect the relative fluorescent intensity even at
50 mol/l (Fig. 6D). These results indicate that PI3K
pathway inhibition suppresses cell-cell contacts
formation and results in decrease of cell number in suspended
Anoikis resistance is a key to the survival of cells in
malignant transformation and metastasis [18,19]. It may also
be a key to the adaptation of cells to suspension culture
and spheroids growth used in engineering. For epithelial
cells, suppression of anoikis upon detachment seems to
be induced when cell-cell contacts are formed. For
example, cadherin-mediated homotypic interactions maintain
the survival of human prostate carcinoma DU-145 cells in
the absence of extracellular matrix (ECM) attachments
. Also, disruption of E-cadherin cell-cell contacts
showed more important for suppressing anoikis of
normal enterocytes after detachment from villus epithelium,
as compared to cell-ECM disruption . Growth as
spheroids renders tumor cells less sensitive to exogenous
apoptotic stimuli, and spheroids have greater drug
resistance than the corresponding monolayers ; and some
cells used in engineering also grow as spheroids in
suspension. Thus, elucidating the mechanisms by which
spheroids survive through cell-cell contacts has
potentially profound value for understanding survival
mechanism of such cells and may also be applied to the control
of growth of cells used in biomedical engineering.
However, much less is known about how survival pathways are
activated under anchorage-independent matrix-deficient
conditions in these cells.
Recently, Marieb et al. found that the adhesion
molecule CD147 promotes anchorage-independent,
hyaluronan-dependent growth of human breast carcinoma cells
. CD147 is also reported to regulate cell-matrix
adhesion . However, it is not clear whether
CD147-mediated cell-cell contact has a role in anoikis resistance or the
growth of spheroids of cells used in engineering. Using
our sophisticated model, which we established from
anoikis-resistant HEK293ar and the parental HEK293 cells,
we found that HAb18G/CD147 cell-cell adhesion
suppresses anoikis in an E-cadherin-dependent manner. In
addition, we have shown that cell-cell adhesion-based
survival signals arising from adjacent HEK292ar cells may
Figure 4 HAb18G/CD147 knockdown reduces the expression of E-cadherin. A. E-cadherin expression and the formation of cell-cell contacts with
time after HAb18G/CD147 siRNA treatment (n = 4-6). Magnification: 400. B. Western blotting to reveal E-cadherin in HAb18G/CD147-siRNA-treated
HEK293ar cells in suspension culture (n = 4-6). -Tubulin was used as a loading control. C. Comparison of the gray scale ratio of E-cadherin/-tubulin
in HAb18G/CD147-siRNA treatment (n = 4-6). ** p < 0.01, siRNA versus scrambled. Each value represents the mean SD of triplicate determinations.
Results are the representative of three similar experiments.
inhibit anoikis in a PI3K/AKT-dependent manner (Figs.
6A, B, C, D). In all, our results indicate that HAb18G/
CD147-mediated cell-cell contacts mediate anoikis
resistance specifically through an E-cadherin-dependent
pathway, and that anoikis suppression mediated by
cellcell contacts in HEK293ar cells involves the PI3K/Akt
Although the data suggest that elevated HAb18G/
CD147 expression is correlated with the progression and
invasion potentials of human hepatoma cells [23,24], the
role of HAb18G/CD147-mediated cell-cell contacts in
acquiring resistance to anoikis remains obscure. Earlier
evidence suggested that CD147 promotes cancer cell
survival by regulating intercellular contacts and inhibiting
anoikis ; in that study, cells transfected with the CD147
gene and expressing different levels of CD147 were used.
In our study, we first found evidence that elevated
endogenous expression of HAb18G/CD147 contributes to
cellcell adhesion and subsequently confers resistance to
anoikis under suspension conditions (Figs. 2A, B, C, E, F).
Our anoikis-resistant HEK293ar cell model would be
more suitable for investigating cell-cell contact-directed
anoikis suppression, since it is closer to the natural in vivo
status of physiological models and cells used in
engineering. In addition, owing to the use of HEK293 cells in
biotechniques, our cell model and the elucidation of the cell
spheroid mechanism may be relevant to bioengineering.
Cell-cell contact-triggered cell survival also involves
anti-apoptotic signalling through E-cadherin-, Src-, and
PI3K/Akt-dependent pathways [15,25]. For instance,
Ecadherin, a classical cadherin that promotes
calciumdependent cell-cell adhesion, suppresses anoikis in
squamous carcinoma and normal proximal tubular cells
[25,26]. In Ewing tumor spheroids, E-cadherin cell-cell
Figure 5 Effect of E-cadherin blocking or knockdown and EDTA on the cell-cell contact formation and HAb18G/CD147. A. The effect of EDTA
and anti-E-cadherin treatment on HAb18G/CD147 expression and cell-cell contacts formation of HEK293ar cells in suspension (n = 4-10).
Magnification: 400. B. The quantification of effect of EDTA and anti-E-cadherin treatment on the cell aggregation (n = 4-10). The degree of cell aggregation
was scored as described in Methods. The cells were counted in 4-10 independent sections (at least 300 nuclei/section). ** p < 0.01, EDTA(0) versus
EDTA(10); ***p = 0.008< 0.01, IgG versus anti E-cadherin. C. Western blotting to reveal E-cadherin expression in HEK293ar cells in E-cadherin RNA
interference (n = 4-6). D. Comparison of the gray scale ratio of E-cadherin/-tubulin in E-cadherin RNA interference (n = 4-6). ** p < 0.01, siRNA versus
scrambled. E. Western blotting of HAb18G/CD147 when E-cadherin was knocked down by E-cadherin siRNA (n = 4-6). p = 0.4775, si RNA versus
scrambled. Each value represents the mean SD of triplicate determinations. Results are the representative of three similar experiments.
Figure 6 The suppression of anoikis in HEK293ar mediated by cell-cell contacts involves the PI3K/Akt pathway. A. The effect of the PI3K
(phosphoinositide 3-kinase) inhibitor LY294002 (20, 50 mol/l) or the ERK (extracellular signal-regulated kinase) inhibitor PD98059 (20, 50 mol/l) on
cellcell contacts formation (n = 4-10). Magnification: 200. B. The effect of signal pathway inhibitors treatment on the degree of cell aggregation (n =
410). The cells were counted in 4-10 independent sections (at least 300 nuclei/section). C. The effect of signal pathway inhibition on the survival of cells
in suspension. The anoikis was determined with TUNEL assay kit. The apoptotic nuclei were counted in 4-10 independent sections (at least 500 nuclei/
section). The concentration of both LY294002 (PI3K inhibitor) and PD98059 (ERK inhibitor) is 50 mol/l. Magnification: 200. D. The effect of signal
pathway inhibitors LY294002 (0, 20, 50 mol/l) or PD98059 (0, 20, 50 mol/l) on cell number. It was evaluated with a CyQUANT NF Cell Proliferation
Assay Kit (C35006, Invitrogen, Ltd) according to the manufacturer's protocol for the nonadherent cells. ** p < 0.01, LY294002 (20) versus LY294002 (50).
Newman-Keuls Multiple Comparison Test. Each value represents the mean SD of at least triplicate determinations. Results are the representative of
three similar experiments.
contacts may activate the ErbB4 RTK signal pathway .
Homophilic E-cadherin binding is also involved in
activating Akt kinase, which ultimately inhibits caspase-3
activity and prevents anoikis . We found that
E-cadherin expression was elevated in the anoikis-resistant
HEK293ar cells (Figs. 3A, B, C) after 24 h suspension
culture. Also, high levels of E-cadherin expression inhibited
anoikis by mediating cell-cell contacts upon cell-matrix
detachment, which was required by HAb18G/CD147,
functioning as an anoikis suppressor. Likewise,
knockdown of HAb18G/CD147 disrupted cell-cell contacts and
anoikis ensued (Figs. 2C, D, E, F). In addition, as
comparison of E-cadherin expression between the HEK293ar and
its parental cell focused on the contrast of this protein
after 24 h suspension culture, this time course should be
long enough for the stabilisation of ligated E-cadherin
and this may preclude the E-cadherin increase from the
stabilisation of ligated E-cadherin at sites of adhesion and
therefore help in valuing the function of this protein
accurately in anoikis resistance. An important conclusion
is that HAb18G/CD147 may have a more prominent role
in suppressing anoikis than E-cadherin, and E-cadherin
may be a downstream effector of CD147 in the
development of anoikis resistance, although the precise
mechanisms by which E-cadherin complexes are remodeled and
degraded remain to be determined.
The pronounced effect of HAb18G/CD147 knockdown
on cell survival may result from a direct decrease in
Ecadherin expression and subsequent loss of cell-cell
contacts (Figs. 4A, B, C). In addition, our more recent data
have shown that LY294002, a specific PI3K inhibitor,
significantly reduced the effect of HAb18G/CD147 on cell
adhesion and metastatic invasion (p < 0.01) of human
hepatoma cells . E-cadherin, predominantly expressed
at cell-cell contacts, is stably bound to the PI3K complex;
this protein expression is necessary and sufficient for
activating the PI3K/AKT pathway . We have also
demonstrated that HAb18G/CD147-mediated cell-cell
contacts can mediate cell survival under
anchorage-independent condition in an E-cadherin-dependent manner;
and that cell-cell contacts mediated resistance of anoikis
involves PI3K pathway in our model. Based on all these
results, we may, at least in part, infer that HAb18G/
CD147 conferred anoikis resistance through E-cadherin
mediated cell-cell contacts, which may activate the PI3K/
AKT pathway and promote spheroids formation by
establishing cell-cell contacts. However, we do not wish
to imply HAb18G/CD147 or E-cadherin stimulates
survival via PI3K stimulation, since this does not occur
directly in response to CD147 and E-cadherin
upregulation in our model. And how HAb18G/CD147 affects
Ecadherin expression needs further investigation.
Using a sophisticated anoikis-resistant model that we
have developed reveals a novel role of HAb18G/CD147 in
cell-cell adhesion leading to anoikis resistance and cell
spheroid growth. Our results support the observation
that HAb18G/CD147 confers anoikis resistance through
E-cadherin-mediated cell-cell contacts; and that cell-cell
mediated resistance of anoikis has been shown to involve
the PI3K pathway. As cell- contact-directed survival is
important for tumor cells in metastasis and invasion,
these results should be relevant to a bitter understanding
of the cell survival mechanism seen in tumor progression.
Furthermore, with the exception of cells of lymphoid
origin, most cultures of mammalian cells used in
bioengineering need to be adapted to suspension conditions of
growth and also capable of forming spheroids in
suspension for survival, these results are also relevant to our
understanding of the survival mechanism of such cells.
Cell lines and tissue culture
The HEK293 cell line was obtained from the Cell Bank of
the Type Culture Collection of the Chinese Academy of
Sciences (Shanghai, China). HEK293ar cells were
acquired in our laboratory and stocked in the CHINA
CENTER FOR TYPE CULTURE COLLECTION
(numbered: CCTCC NO: C200927). Cells were cultured in
RPMI 1640 supplemented with 10% heat-inactivated fetal
bovine serum (FBS) at 37C in a humidified atmosphere
containing 5% CO2. Methocel medium consisted of RPMI
1640 supplemented with 0.8% methocel and 10% FBS.
The cell detachment culture was a modification of the
suspension culture procedure previously described .
Briefly, flasks were coated with 1.5-2.0% sterilized agar
and supplemented with RPMI 1640/10% FBS. Every 2-3
days, the culture flask was refed regularly by carefully
removing the old medium and adding 10-15 ml fresh
methocel medium. All cultures were monitored routinely
and to ensure they were free of mycoplasma, fungal and
bacterial contamination. All cell lines were discarded
after two months and new lines were propagated from
A small interfering RNA (siRNA) targeting E-cadherin
(Genbank accession No. NM_004360) was generated:
siRNAE-cad 5'-CAGACAAAGACCAGGACUA-3' .
(5'-GUUCUUCGUGAGUUCCUCdTdT-3', 3'-dTdTCAAGAGCA CUCAAGGAG-5')
 and siE-cadherin-cad were synthesized by Ambion,
Inc. Cells were transfected with siRNA using Lipofectin
2000 (Invitrogen, Ltd, USA), according to the
manufacturer's instructions. Briefly, HEK293ar cells in
exponential growth phase were transfected with the
corresponding targeted siRNA (100 nmol/l) or a
scrambled RNA, or mock-treated with Lipofectin 2000,
serumand antibiotic-free RPMI 1640 medium (control).
Transiently transfected cells were grown for 24 h and re-plated
on agar-coated six-well plates. The cells were incubated
for a further 24 h before the experiments were conducted.
Silencer negative control siRNA (Ambion, USA) was used
under similar conditions as a negative control.
The morphology of apoptotic cells was assessed by
phase-contrast (Olympus, Japan) and electron
microscopy (EM). Briefly, cells were cultured under cell
detachment conditions for the indicated times, then observed
and photographed. To evaluate the subcellular
morphology characteristic of apoptosis, transmission electron
microscopy (TEM, JEM-2000EX, JEOL, Japan) and
scanning electron microscopy (SEM, S3400 N, HITACHI,
Japan) were used, both by a previously-described method
. Each sample was viewed in 4-10 independent
sections (at least 100 cells/section).
To detect the fragmented DNA of apoptotic cells, a
DeadENDEM Colorimetric TUNEL System (Promega,
USA) was used, according to the manufacturer's protocol.
Cells from detached and attached cultures were scored
for incidence of TUNEL (+) staining using phase-contrast
microscopy. Apoptotic nuclei (TUNEL) with condensed
chromatin were darkly stained.
DNA fragmentation was also quantified by flow
cytometry (Becton Dickinson, USA) using a DNA PREP
Reagents kit (Bechman, Coulter, Inc., USA). Fluorescence
intensity of propidium iodide (PI) was measured at 670
nm (FL3-H). In the histogram, the y-axis is the number of
cells counted (labelled Counts); the x-axis is the DNA size
and content for each cell registered (labelled FL3-H).
Cells with normal DNA have more intense fluorescence.
Apoptotic cells, which have more fragmented DNA, have
lower fluorescence intensity. The sub-G1 region (left side
of the normal peak) is apoptotic. The number of cells in
the sub-G1 region divided by the total cell count indicates
the percentage apoptosis. Each sample with a minimum
of 1/104 cells was examined at least three times. Gating of
the side-scatter plot excluded debris and gating of the
pulse width plot excluded any remaining cell aggregates
Cell-cell contact s perturbation
Formation of cell-cell contacts was disturbed using a
calcium chelator or E-cadherin antibody. Briefly, cells
seeded at 2 105/ml in agar-coated six-well plates were
treated with 0 or 10 mmol/l EDTA for 12 h at 37C.
Alternatively, cells were treated with 10 g/ml mouse
anti-Ecadherin (Abcam Ltd. Cambridge, UK), with the same
amount of goat IgG (Sigma, USA) being used as control.
The degree of cell aggregation was scored as follows; no
aggregation (-), 1-5 cells/aggregate (1+), 6-10
cells/aggregate (2+), 10-15 cells/aggregate (3+), greater than 15
cells/aggregate (4+). Photographs were taken with an
Olympus camera under an inverted microscope .
Cells were harvested in a lysis buffer, and total protein
quantified using BCA. Equal amounts (20 g) of protein
were subjected to 8% sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) under
non-reducing conditions. The separated proteins were transferred
to polyvinylidene difluoride (PVDF) membranes
(Millipore, Bedford, MA), which were immunoblotted with the
appropriate primary antibody. The following primary
antibodies were used: human hepatoma monoclonal
HAb18 (1:5000, prepared in our laboratory) and
anti-Ecadherin mouse monoclonal (1:800; Abcam, Ltd.
Cambridge, UK). HRP-conjugated rabbit-anti-mouse
immunoglobulin (1:5000, Pierce, Rockford, IL) was used as the
secondary antibody. -Tubulin (Abcam Ltd. Cambridge,
UK) was used as loading control. As for HAb18G/CD147,
two major forms of HAb18G/CD147 (43-66 and 35 kDa)
were analyzed as previously described . The
immunoreactive bands were visualized using a chemiluminescent
substrate detection system (GE, Healthcare, USA). The
Ecadherin western blotting was done as previously
described . Quantification of bands from two similar
experiments was done using Gene Tool Image software
(n = 4-6).
Cells were harvested and dried on coverslips, fixed with
ice-acetone for 15 min at 4C. The fixed cells were
blocked with 5% non-fat milk for 1 h before being
incubated with HAb18 mAb (1:200) and anti-E-cadherin
mouse monoclonal antibody (1:50; Abcam, Ltd.
Cambridge, UK) in blocking solution for 2 h and stained with
FITC-conjugated anti-mouse IgG (1:1000, Pierce,
Rockford) for 1 h. Finally, the nuclei were stained with 100 ng/
ml DAPI (4', 6-diamidino-2-phenylindole; Biotium,
Hayward, USA) in PBS for 3 min. The stained cells were
examined with a laser scanning confocal microscope
(Olympus FV1000, Tokyo, Japan). Fluorescence density of
the confocal images was measured by Image Pro Plus 6.0
Treatment with signal transduction inhibitors
HEK293ar cells were treated with the ERK inhibitor
PD98059 (0-50 mol/l; Sigma, USA), the PI3K inhibitor
LY294002 (0-50 mol/l; Sigma, USA), or the DMSO
vehicle as a control for 2 h under adhesion conditions. The
treated cells were cultured under cell-detachment
conditions for the indicated times. Cell-cell contacts were
observed with an inverted phase-contrast microscope
and cell number was evaluated with a CyQUANT NF
Cell Proliferation Assay Kit (C35006, Invitrogen, Ltd,
USA), which is based on measurement of cellular DNA
content via fluorescent dye binding. The fluorescence
intensity of each sample was measured using a
fluorescence microplate reader with excitation at ~485 nm and
emission detection at ~530 nm. Experimental protocols
of assay were done according to the manufacturer's
instructions for the nonadherent cells.
All data were expressed as mean SD from at least three
independent experiments. Statistical analysis was
performed by Student's t-test or Newman-Keuls Multiple
Comparison Test, using SPSS 11.5 statistical software. All
statistical tests were two-sided and p values < 0.05 were
considered statistically significant.
XKM and LW carried out the experimental protocols, participated in the design
of the experiments, performed experiments, and drafting for manuscript. YL,
PZ, HT and XMY collected microscope images of anoikis-resistant cells, and
helped draft the manuscript. PZ, LL, and ZNC conceived the study, participated
in its design and coordination, and were also involved in drafting the
manuscript. All authors read and approved the final manuscript.
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